Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

Summary

Infectious pneumonia is among the most common infections in human. An appropriate in vivo model is critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics. With this murine oropharyngeal aspiration pneumonia model, one can examine the pathogenesis and new treatments against these deadly infections.

Abstract

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative pathogens. Infectious pneumonia is among the most common infections presented by patients in the clinic and thus warrants an appropriate in vivo model. Typical pneumonia models use intranasal inoculation, which deposits excessive organisms outside the lung, causing off-target complications and symptoms, such as sinusitis, gastritis, enteritis, physical trauma, or microparticle misting to mimic aerosol spread more typical of viral, tuberculous, or fungal pneumonia. These models do not accurately reflect the pathogenesis of typical community- or healthcare-acquired bacterial pneumonia. In contrast, this murine model of oropharyngeal aspiration pneumonia mimics the droplet route in healthcare-acquired pneumonia. Inoculating 50 µL of the bacteria suspension into the oropharynx of anesthetized mice causes reflexive aspiration, which results in pneumonia. With this model, one can examine the pathogenesis of pneumonia-causing pathogens and new treatments to combat these diseases.

Introduction

Lower respiratory infection is the world's deadliest communicable disease and the most common cause of death in developing countries¹. Globally, these infections account for more than 3.2 million deaths¹. In addition, nosocomial pneumonia is among the most common and deadly forms of healthcare acquired infections, and is caused by the most antibiotic-resistant pathogens^2,3. The typical route of acquisition of bacterial pneumonia for both community-acquired and nosocomial pneumonia is the aspiration of oropharyngeal contents into the alveoli. Murine models use....

Protocol

All procedures involving animals must be approved by the researcher’s Institutional Animal Care and Use Committee (IACUC).

1. Preparation of Bacterial Inoculum

1. Isolate bacterial colonies.
   1. Streak a bacterial strain (e.g., A. baumannii “HUMC1”) on appropriate sterile agar medium (e.g., Tryptic soy agar), being careful to generate isolated colonies.
   2. Incubate at appropriate conditions (e.g., overnight at 37 °C).
2. Grow overnight culture.
   1. Select a representative, isolated colony from the agar plate with a sterile inocul....

Results

By carefully following the protocol, reproducible and robust data can be easily obtained. It is critical to strictly adhere to one's customized inoculum preparation protocol for experiments to be compared to one each other. It is also important to properly handle mice during the infection procedure. Be sure to place mice into an anesthesia chamber devoid of isoflurane. Mice will panic if they are placed into a chamber that has been pre-filled with isoflurane and may experience excess stre.......

Discussion

To be sure, mice are not miniature humans. Results obtained from mouse models must be considered in context and subsequently interpreted for applicability to humans, based on differences and similarities between the two species⁶. It is also important to choose the appropriate mouse strain as certain are more susceptible to some infections than others; the same applies to the pathogen strain of choice¹⁶.

It is essential to perform infections in an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	BD	214530	Combine with TSB to make TSA
Beads, Borosilicate Glass	Kimble	135003	Sterilize by baking or autoclaving before each use
Beaker, 250 mL	Pyrex	1003	Used during precise aliquoting of concentrated bacterial inocula
Centrifuge	Sorvall	ST 40R	Capable of 4,000×g at 4°C
Chamber for Anesthesia	Kent Scientific Corporation	VetFlo-0720	Accommodates up to 5 mice
Cryomold, Intermediate Size	Sakura Tissue-Tek	4566	Disposable vinyl specimen molds, 15×15×5 mm
Dental Floss	Oral-B	37000469537	Tie to stable post approx. 6" above table height
Forceps	VWR	82027-440	Used to gently pull tongue out of mouse's mouth
Homogenizer for Lung Tissue	Omni International	TM125-115	Autoclave before first use; rinse between samples
Isoflurane for Anesthesia	Abbott	10015516	Alternative drug can be used; modify procedure accordingly
iSTAT Cartridge	Abbott	03P79-25	Various cartridges are available to suit your needs
Ketamine, 100 mg/mL	Western Medical Supply	4165	Dilute 1:10 in PBS to 1 mg/mL and combine with Xylazine at 1 mg/mL
Ointment for Eyes	Akorn	Tears Renewed	Avoid touching eye with tip of dispenser
Optimal Cutting Temperature (O.C.T.) Compound	Fisher Scientific	23-730-571	Used to freeze lung samples at -80 °C to prepare for pathology sectioning
Petri Dish	VWR	25384-302	Polystyrene, disposable, sterilized, 100×15 mm
Phosphate-Buffered Saline (PBS)	Corning	21-031-CM	Dulbecco's PBS without calcium and magnesium
Pipette Tips, 200-μL	VWR	10017-044	Autoclave before use
Pipetter, 200-μL	Gilson	Pipetman P200	Autoclave and calibrate before use
Spreader, Bacterial Cell	Bel-Art	F377360006	Sterilize by baking or autoclaving before each use
Stir Bar, Magnetic, 7.9 mm Diameter × 38.1 mm Length	VWR	58948-150	Used for stiring concentrated bacterial inocula during aliquoting
Stir Plate, Magnetic	Corning	PC-620D	Used for stiring concentrated bacterial inocula during aliquoting
Tryptic Soy Broth (TSB)	BD	211822	Combine with Agar to make TSA
Vial, Conical, Sterile, 50 mL	Corning	431720	Used for preparing bacterial inocula
Vial, Conical, Sterile, 500 mL	Corning	431123	Used to concentrate inocula for preparing frozen inocula
Vial, Cryogenic, 2.0 mL	Corning	430659	Used for cryogenic storage of concentrated bacterial inocula
Xylazine, 20 mg/mL	Akorn	AnaSed Injection	Dilute 1:20 in PBS to 1 mg/mL and combine with Ketamine at 1 mg/mL